Download Chapter 1 - bYTEBoss

Chapter 14
Forensic DNA Typing
Objectives
• Students should gain an understanding of:
– The use of the polymerase chain reaction
(PCR) to make many copies of a DNA
sequence
– Short tandem repeats (STRs) and their
forensic importance
– The use of electrophoresis to analyze STRs
– The Combined DNA Index System (CODIS)
– DNA paternity testing
– Mitochondrial DNA testing
Introduction
• The DNA in all cells of an individual is the
same through the body.
• DNA contains repeated sequences of
genetic codes with core sequences that
are unique to particular individuals.
• The genetic code can be determined from
a small amount of DNA.
Restriction Fragment Length
Polymorphisms (1 of 3)
• DNA contains genes that control production
of proteins in the body.
• Other sections act as spacers between the
coding areas.
– The sequences of bases in the noncoding
regions are used for DNA profiling.
– The sequences vary greatly from person to
person.
Restriction Fragment Length
Polymorphisms (2 of 3)
• RFLP allows for the individualization of
DNA evidence
– Step 1: DNA is extracted from a chromosome
– Step 2: restriction enzymes cut the DNA
strands into fragments at specific base
sequences
Restriction Fragment Length
Polymorphisms (3 of 3)
• Disadvantages of RFLP
– Takes 6–8 weeks to obtain results
– Requires a large sample of intact, nondegraded
DNA
– Not amenable to high-volume sample processing
– Produces very large DNA strands that are often
damaged when the recovered DNA is partially
degraded
Polymerase Chain Reaction: A
DNA Copy Machine (1 of 3)
• Advantages of PCR
– Allows many copies of a portion of DNA
sequence to be manufactured in the DNA lab
– Amplifies only those DNA regions that are of
interest
– Is fast and extremely sensitive
Polymerase Chain Reaction: A
DNA Copy Machine (2 of 3)
• Thermocycling: DNA is repeatedly heated
and cooled
– 194 °F: the two complementary DNA strands
separate
– 140 °F: each primer finds and binds to its
complementary sequence on the DNA strand
– 162 °F: the DNA polymerase enzyme adds bases
to extend the primer and to build a DNA strand
that is complementary to the sample DNA
Polymerase Chain Reaction: A
DNA Copy Machine (3 of 3)
• The thermocycling process is repeated to
make more copies.
• Each heating–cooling cycle doubles the
number of copies.
• DNA laboratory and technicians must take
extreme care to eliminate extraneous DNA
from the PCR amplification area.
Short Tandem Repeats
• STRs: locations on the sample DNA that
contain a short sequence of bases that is
repeated over and over
– Four-base repeats are typically used for forensic
purposes.
– STRs are often recovered from bodies or stains
that have started to decompose.
– They can be amplified very quickly.
DNA Sequence Variations among
Individuals
• Individuals differ genetically because they
possess different combinations of alleles at
numerous locations in their genomes.
• Only 3% of a person’s DNA is involved in
coding for proteins.
• Mutations in noncoding regions have no
effect on the phenotype of a person.
• Loci selected for DNA typing are selectively
neutral; they confer neither benefit nor harm
to the individual’s ability to reproduce.
Inheritance of Alleles
• Alleles are inherited from an individual’s
parents following the fundamental rules of
genetics.
• Different individuals posses different alleles
in numerous loci in their genomes.
• Investigators measure the length of STRs at
different locations to determine a person’s
genetic identity.
Analyzing the STR by
Electrophoresis (1 of 3)
• Electrophoresis
– Causes ions in solution to migrate under the
influence of an electric field
– Separates STRs according to their length:
smaller DNA molecules move faster
– Establishes the number of repeats and
elucidates the genotype of the individual at each
amplified locus
Analyzing the STR by
Electrophoresis (2 of 3)
• Gel electrophoresis
– After voltage is applied 2–3 hours,
electrophoresis stops and the DNA is made
visible.
– Each group of similar-length molecules
appears as a narrow band in the gel.
– By comparing the locations of the bands in
each sample lane to the ladder, the technician
can determine the STR type for each sample.
– Gel electrophoresis is slow, is difficult to
automate, and can be dangerous.
Analyzing the STR by
Electrophoresis (3 of 3)
• Capillary electrophoresis
– Allows for greater heating than is possible with
a slab gel
– Uses a higher voltage, so molecules migrate
much faster
– Produces high-speed, high-resolution
separations on extremely small samples
– Uses laser fluorescence: fluorescent dye is
attached to the PCR primer that amplifies the
STR region of interest
Multiplex DNA Analysis (1 of 4)
• Multiple STR loci may undergo PCR
amplification and be analyzed
simultaneously.
• Multiplexing is accomplished by placing each
of the four dyes on specific primers and by
adjusting the size of the STR amplicon
produced.
Multiplex DNA Analysis (2 of 4)
• Multiplexing by size
– Amplicons from different loci that are different
sizes are clearly separated from one another
and appear at different locations on the x-axis in
the CE analysis.
– It isn’t possible to multiplex more than five or six
loci.
Multiplex DNA Analysis (3 of 4)
• Multiplexing by dye color
– Different dyes amplify STR loci that are the
same size and cannot be separated using CE.
Multiplex DNA Analysis (4 of 4)
• Multiplexing with multiple capillaries:
capillary array electrophoresis
– Parallel capillaries lie next to one another and
process multiple samples simultaneously
– Technique reduces the time between when a
DNA sample is collected at the crime scene
and when it is actually analyzed
Forensic STRs
• Most DNA databases rely on 10 or more STR
loci, each of which is found on a different
chromosome.
• Standard nomenclature is used to designate
the location of a DNA marker.
– If the marker is part of a gene or falls within it,
the gene name is used.
– If the STR falls outside a gene region, its name
indicates the chromosome and locus on which it
is found.
CODIS (1 of 4)
• Combined DNA Index System
– Created in 1994 as part of the DNA
Identification Act
– Consists of a national database containing the
DNA of individuals convicted of sexual and
violent crimes
– Assisted in 20,000 investigations in 2004
CODIS (2 of 4)
• Three tiers of CODIS
– Local: labs maintain a local DNA index
– State: combines the profiles of all local labs
– National: compares profiles of all state systems
CODIS (3 of 4)
• All 50 states maintain databases for
sexual offenders and convicted murderers
• 49 states include violent felons
• 43 states include all felons
CODIS (4 of 4)
• Use of CODIS
– The computer compares the DNA profile
submitted with profiles on file in the network.
– If a match is found in the Convicted Offender
Index, the lab is sent the identity of the
perpetrator.
– If a match is found in the Forensic Index, two
crimes have been linked together. The labs must
then verify the match; law enforcement may then
pool resources to solve the crimes.
Interpretation of DNA Profiles
(1 of 3)
• It is easier to use DNA to exclude a person
from suspicion than to prove that the person
is the only suspect.
• The Innocence Project reports that three
times more suspects are proven innocent by
DNA analysis than are proven guilty.
• The loci used for DNA matches must be
chosen to minimize the chance that two
people will have the same profile.
Interpretation of DNA Profiles
(2 of 3)
• Hardy–Weinberg principle: allele frequencies
remain constant from generation to
generation and allele frequencies can be
easily calculated
– Frequency of a particular homozygote = allele
frequency squared
– Expected frequency for a heterozygote = 2 × the
product of the two allele frequencies
Interpretation of DNA Profiles
(3 of 3)
• Interpreting multiple DNA profiles:
– Prevalence of a particular CODIS profile in the
general population: multiply the genotype
frequencies for all the loci together
– Likelihood ratio: compares the probabilities of
alternative events
• The true discriminating power of CODIS is
achieved by multiplying the individual
frequencies of the 13 loci.
Paternity Testing (1 of 2)
• A child can receive only one of the father’s
alleles and one of the mother’s alleles.
• A familial pattern should be obvious by
comparing the DNA profiles of mother,
father, and child.
Paternity Testing (2 of 2)
• Paternity index: the likelihood that an allele
from the child supports the assumption
that the tested man is the true biological
father
• Combined paternity index: determined by
multiplying the individual PIs for each
locus tested
Mitochondrial DNA Analysis (1 of 5)
• Examination of recovered mitochondrial
DNA is useful in circumstances of badly
decomposed or burned bodies, old bones,
and human hair without follicular tags
• mtDNA is rarely used in criminal
proceedings.
• It has been useful for historical
investigations.
Mitochondrial DNA Analysis (2 of 5)
• Mitochondrial DNA (mtDNA)
– Is a circular DNA molecule that is only 16,569
pairs in circumference
– Has no noncoding elements; every base has
a function
– For the most part, is the same in all
individuals
Mitochondrial DNA Analysis (3 of 5)
• Variations in mtDNA
– The D-loop contains two hypervariable regions
whose sequences vary.
– A difference of less than 3% is expected
between unrelated individuals.
Mitochondrial DNA Analysis (4 of 5)
• DNA sequencing: determines the
sequence of bases along a DNA strand
• Anderson sequence: the first mtDNA
hypervariable sequence to be determined;
serves as a reference sample
Mitochondrial DNA Analysis (5 of 5)
• mtDNA is inherited from a person’s mother.
• All brothers and sisters of the same mother
have the same mtDNA, which is also the
same as the mtDNA of their maternal
grandmother and their mother’s siblings.
The Y Chromosome: STRs and
SNPs (1 of 2)
• STR analysis is directed at the Y
chromosome.
• The Y chromosome contains polymorphisms
that might eventually be used as forensic
markers.
The Y Chromosome: STRs and
SNPs (2 of 2)
• Single-nucleotide polymorphisms (SNPs)
(2 of 2)
– The base difference occurs at only one
specific site.
– SNPs at different loci can be determined
simultaneously, producing an SNP DNA
profile.
Low-Copy-Number DNA Typing
• Applications
– Used when the quantifying test indicates too little
DNA is available to perform a regular DNA
analysis
– Used only in cases where standard typing
protocols have already failed
• As the amount of suspect DNA decreases,
the chance of contamination by DNA from
other sources increases.